Process for producing itaconic acid under anaerobic conditions
an anaerobic and process technology, applied in the direction of biochemical equipment and processes, fermentation, lyse, etc., can solve the problems of limited yield of aerobic processes and reduced product yield, and achieve the effect of enhancing the availability of precursors
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example 1
ous Expression of cadA, acnA, and gltA
[0111]Gene cadA from A. terreus was codon optimized and expressed in E. coli to enable itaconate production. Small amounts of itaconate were produced (below 10 mg / L) when E. coli BW25113 (DE3) (pKV-C) was cultivated in LB in shake flask cultures at 37° C., but no detectable CadA activity was found in CFE of these cultures. SDS-PAGE analysis showed that almost all CadA was present in the form of inclusion bodies (data not shown). As inclusion bodies are often associated with fast and high-level expression of heterologous proteins (Jurgen et al. 2010 Microbial Cell Factories 9(1)), two measures were taken to reduce these rates: cultivation in MM instead of LB and cultivation at lower temperatures. When E. coli BW25113 (DE3) (pKV-C) was grown in MM in pH-controlled bioreactors at 37°, CadA could be detected in CFE with a specific activity of 0.03 U / mg. The activity was further increased to 0.38 U / mg when the cultivation temperature was lowered to 3...
example 4
Δpta-ΔldhA on Growth Under Anaerobic Conditions
[0118]E. coli BW25113 (DE3) (pEV) and E. coli BW25113 (DE3) Δpta-ΔldhA (pEV) were grown on MM in pH-controlled bioreactors under anaerobic conditions with glucose as carbon source. The main fermentation products of E. coli BW25113 (DE3) (pEV) were lactate, ethanol, formate and acetate (FIG. 5), which accounted for 74% of the carbon that was added to the culture (Table 3). As a lot of formate (16% Cmol) was formed in E. coli BW25113 (DE3) (pEV), only low amounts of CO2(3 mM / L, 2 (12 mM / L) were produced. The formation of acetate was redox balanced with the co-production of ethanol and succinate.
[0119]E. coli BW25113 (DE3) Δpta-ΔldhA (pEV), in which pta, encoding phosphate acetyltransferase, and ldhA, encoding lactate dehydrogenase were eliminated, still produced acetate in comparable amounts as E. coli BW25113 (DE3) (pEV), but lactate was no longer formed (FIG. 6; Table 3). E. coli BW25113 (DE3) Δpta-ΔldhA (pEV) did not produce formate. I...
example 5
Production Under Anaerobic Conditions
[0120]E. coli BW25113 (DE3) and E. coli BW25113 (DE3) Δpta-ΔldhA containing pKV-C or pKV-CGA were grown on MM in pH-controlled bioreactors under anaerobic conditions. pKV-C and pKV-CGA both express codon-optimized cadA, which encodes the cis-aconitate decarboxylase from Aspergillus terreus that was previously shown to enable the production of itaconate in E. coli. pKV-CGA also expresses citrate synthase (gltA) and aconitase (acnA) from Corynebacterium glutamicum. These genes enhanced the production of itaconate in E. coli BW25113 (DE3) Δpta-ΔldhA under aerobic conditions (see Examples 1 to 4).
[0121]Expression of cadA did not result in itaconate production in E. coli BW25113 (DE3) (pKV-C) (FIG. 5), but 0.08 mM of itaconate was produced by E. coli BW25113 (DE3) Δpta-ΔldhA (pKV-C) (FIG. 6). A similar amount of itaconate was formed by E. coli BW25113 (DE3) (pKV-CGA) (FIG. 5). E. coli BW25113 (DE3) Δpta-ΔldhA (pKV-CGA) produced eight times more itacon...
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